Immunohistochemical Techniques for Tissue Sections

As previously described, tissues and organoids were fixed in 10% formalin, paraffin embedded, and sectioned.^{23 (link),52 (link)} Antigen retrieval was performed with Borg Decloaker RTU solution (Biocare Medical) in a pressurized Decloaking Chamber (Biocare Medical) for 3 minutes. The following antibodies were used: mouse monoclonal β-catenin (1:200, BD Biosciences 610154), rabbit monoclonal CDX2 (1:1250, Abcam ab76541), rabbit monoclonal human specific Cytokeratin 20 (1:500, Abcam, ab76126), rabbit polyclonal Lyve 1 (1:200, Abcam, ab14917), rabbit monoclonal Anti-CD3 (1:200, Abcam ab16669), CD31 (1:50, Abcam, ab28364), and the human specific nuclear envelope marker Lamin A+C (1:2500, Abcam, ab108595). Biotin-conjugated secondary donkey anti-rabbit or anti-rat antibodies were used from Jackson ImmunoResearch. The Vectastain Elite ABC immunoperoxidase detection kit (Vector Labs PK-6101) followed by Dako Liquid DAB+ Substrate (Dako) was used for visualization. Hematoxylin and eosin (H&E) and trichrome stains were performed according to standard procedures.

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Roper J., Tammela T., Cetinbas N.M., Akkad A., Roghanian A., Rickelt S., Almeqdadi M., Wu K., Oberli M., Sánchez-Rivera F., Park Y., Liang X., Eng G., Taylor M.S., Azimi R., Kedrin D., Neupane R., Beyaz S., Sicinska E.T., Suarez Y., Yoo J., Chen L., Zukerberg L., Katajisto P., Deshpande V., Bass A., Tsichlis P.N., Lees J., Langer R., Hynes R.O., Chen J., Bhutkar A.J., Jacks T, & Yilmaz Ö.H. (2017). In vivo genome editing and organoid transplantation models of colorectal cancer. Nature biotechnology, 35(6), 569-576.

Publication 2017

Borg Decloaker RTU solution Decloaking Chamber ab76541 ab76126 ab14917 ab16669 ab28364 ab108595 Biotin-conjugated secondary donkey anti-rabbit or anti-rat antibodies Vectastain Elite ABC immunoperoxidase detection kit Dako Liquid DAB+ Substrate

Anti antibodies Anti cd3 Antibodies Antigen Biotin Cytokeratin 20 Donkey Eosin Formalin Hematoxylin Human Immunoperoxidase Lamin a c Mouse Nuclear envelope Organoids Paraffin Rabbit Rtu solution Tissues Trichrome stains Vector β catenin

Corresponding Organization :

Other organizations : Allen Institute, Massachusetts General Hospital, Dana-Farber Cancer Institute, Tufts Medical Center, Molecular Oncology (United States), University of Helsinki, Howard Hughes Medical Institute, Massachusetts Institute of Technology

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Variable analysis

independent variables

Antigen retrieval with Borg Decloaker RTU solution in a pressurized Decloaking Chamber for 3 minutes

dependent variables

Immunostaining results for the following markers: β-catenin, CDX2, Cytokeratin 20, Lyve 1, CD3, CD31, Lamin A+C
Hematoxylin and eosin (H&E) and trichrome staining patterns

control variables

Tissue and organoid samples were fixed in 10% formalin, paraffin embedded, and sectioned

controls

No positive or negative controls were explicitly mentioned in the provided information

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